title: Fluorescence polarization study of a salt bridge between a single chain Fv and its antigen ribonuclease A.
abstract: The interaction between a single chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6) M(-1) in PBS at pH 7.2 and 37 degrees C, respectively. Whereas the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan. katakura@bio.eng.osaka-u.ac.jp
authors.forename: Y
authors.initials: Y
authors.lastname: Katakura
authors.forename: T
authors.initials: T
authors.lastname: Kumamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Iwai
authors.forename: Y
authors.initials: Y
authors.lastname: Kurokawa
authors.forename: T
authors.initials: T
authors.lastname: Omasa
authors.forename: K
authors.initials: K
authors.lastname: Suga
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D012965:Sodium Chloride; D012259:Ribonuclease, Pancreatic